We proposed that an equilibrium exists between 1) nucleoside RT inhibitor (NRTI) incorporation and degradation of the RNA template by RNase H activity, and 2) NRTI excision and resumption of DNA synthesis. Degradation of template RNA leads to dissociation of the template:primer and termination of DNA synthesis unless the NRTI is excised and DNA synthesis is resumed. Studies of RNase H mutants provided strong evidence in support of this hypothesis and novel insights into the mechanism of zidovudine (AZT) resistance and NRTI-mediated abrogation of viral DNA synthesis. We carried out genotypic and phenotypic analyses of patient-derived C-terminal domains of RT for resistance-associated mutations, and identified eight novel mutations in the cn domain of RT that substantially enhance AZT resistance when the polymerase (pol) domain contains thymidine analog resistance mutations (TAMs). We performed biochemical and genetic analyses to determine the mechanism by which the cn domain mutations enhance AZT resistance. We are currently analyzing additional subtype B-infected adult and pediatric patient samples as well as subtype C-infected patient samples for cn domain mutations associated with NRTI and NNRTI resistance. We will determine the role of C-terminal domains in conferring antiviral drug resistance to subtype C and other non-B subtypes. [Corresponds to Pathak Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]